The present invention relates to fluorescence-based assays for measuring enzyme activity, particularly cleaving and joining reactions.
Assays for measuring enzyme activity, particularly enzyme cleaving activity such as hydrolysis, are widely employed in the biological and pharmaceutical sciences. With the advent of combinatorial chemistry and high throughput screening, there is a growing need for simple, sensitive and cost-effective assays to screen for potential modulators of enzyme activity. Of particular interest to the pharmaceutical industries are methods for detecting proteolytic enzyme cleavage.
Fluorescence-based assays offer significant advantages over radiochemical, ELISA, antibody and more traditional techniques for measuring enzyme cleaving activity in terms of simplicity of handling, sensitivity, cost and ease of automation. Thus, for example, hydrolysable fluorescent substrates are known in the art which, when cleaved, provide fluorescent dyes (EP 0231125) which can be used to measure enzyme activity. Similar substrates are reported in EP 0004256 for use in determining protease activity following proteolytic release of fluorogenic groups. Peptides which are intramolecularly quenched by virtue of synthetic quenching groups (e.g. DABCYL) have been disclosed which have utility as imaging probes (e.g. WO 02/056670). Whilst such fluorogenic labels may provide an effective means of determining enzyme activity they are generally detectable at long wavelengths, typically in the region of 500-600 nm.
More recently there has been considerable interest in the application of fluorescence resonance energy transfer (FRET) assays which involve the use of substrates having donor and quenching acceptors on the same molecule. WO 94/28166 reports the use of such FRET labels attached to a polypeptide substrate which fluoresce more intensely on hydrolysis by a protease. A similar principle is employed in the fluorogenic substrates disclosed in EP 0428000 wherein the peptide substrate has a viral protease enzyme-cleavable site. U.S. Pat. No. 5,708,137 also discloses the use of a fluorogenic substrates to detect viral proteases which comprise internal donor and acceptor/quenching groups.
Methods for fluorescently labelling and quenching peptides have also recently been disclosed. Thus WO 02/081509 describes the use of tryptophan, tyrosine or histidine residues to internally quench fluorescence intensity within fluorescently labelled peptides. The peptides can be used to detect endo- and exo-peptidase activity.
While FRET techniques offer greater sensitivity and reliability for use in screening assays than simple fluorescent intensity techniques, the substrates are considerably more expensive to prepare and purify due to their complex nature. Thus the preparation of FRET labels is demanding in terms of both analytical and/or purification and material costs. Furthermore the only method for distinguishing conventional fluorescent or FRET labels is by their absorption and emission spectra.
Fluorescence lifetime measurements that may be utilised in the present invention offer significant advantages over conventional fluorescence techniques that are based solely on quantifying fluorescence intensity. Fluorescence lifetime is determined from the same spectrally resolved intensity signal, but is additionally resolved in the temporal domain. Fluorescence lifetime techniques provide greater discrimination because the signal is largely unaffected by ‘background noise’. A further advantage with this technique is that several different events can be measured simultaneously by selecting labels having distinguishable lifetimes, thus enabling multiplexing. In addition, measurements of fluorescence lifetime are unaffected by concentration effects and photobleaching.
There is therefore a continued need in the biological and pharmaceutical sciences for improved fluorescence-based assays for measuring enzyme cleaving activity. Such assays may have one or more of the following attributes: high sensitivity, good reliability, robustness, simplicity of use, low cost, ease of automation, label specificity and/or more than one form of detection for distinguishing labelled compounds. Preferably the improved assays display more than one of these features and preferably they display all of these features.
It is thus an object of the invention to provide a method of measuring the activity of an enzyme in cleaving a substrate comprising a single fluorescent label and an enzyme cleavable linkage group. It is also an object of the invention to provide a method of screening agents which affect or modulate enzyme cleaving activity. It is a further object of the invention to provide a method of measuring cellular location and distribution of a labelled substrate.
A further object of the present invention is to provide a method for measuring the activity of an enzyme to join a substrate to a reactant.